Novel derivatives of beta-endorphin, intermediates therefor and compositions and methods employing said derivatives

ABSTRACT

Novel hentriacontapeptides having the following amino acid sequence: 
     
         H-Tyr-X-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys 
    
     -Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu-Y 
     wherein X is a chiral residue of a D-amino acid selected from the group consisting of D-alanine, D-leucine, D-isoleucine, D-valine, D-phenylalanine, D-tyrosine, D-tryptophan, D-serine, D-threonine, D-methionine, D-glutamic acid, D-glutamine, D-aspartic acid, D-asparagine, D-lysine, D-proline, D-histidine or D-arginine; Y is selected from the group consisting of hydroxy, amino, loweralkylamino, diloweralkylamino and lower alkoxy; and the pharmaceutically acceptable salts thereof; intermediates useful in making the novel compounds and pharmaceutical compositions and methods employing the novel compounds.

BACKGROUND OF THE INVENTION

While there are a number of analgesic agents currently utilized torelieve mild to severe pain, the search for improved analgesics is acontinuing one because of the numerous problems associated with thepresently available agents. Aspirin and related salicylates areconsidered to be non-narcotic analgesic agents useful for relieving mildto moderate pain in addition to their usefulness as anti-inflammatoryand anti-pyretic agents. However, the ingestion of salicylic acid orrelated salicylates may result in epigastric distress, nausea andvomiting. This widely used class of non-narcotic analgesic agents mayalso cause gastric ulceration and even hemorrhage both in experimentalanimals and man. Exacerbation of peptic ulcer symptoms and erosivegastritis have all been reported in patients on high dose therapy, i.e.,arthritis patients. Aspirin is also one of the most common causes ofdrug poisoning in young children and has a potential of serious toxicityif used improperly.

Acetaminophen is also considered to be a non-narcotic analgesic agentuseful in treating mild pain associated with simple headache, commonmuscular aches, etc. While acetaminophen is particularly useful forpatients who cannot take aspirin, i.e., ulcer patients, its use iscontraindicated in individuals who have exhibited a sensitivity to it.In addition to their drawbacks in view of their potential side effects,the mild nonnarcotic analgesic agents are not sufficiently potent torelieve the severe pain associated with surgery, cancer and the like.

Unfortunately, the potent analgesic agents capable of relieving suchsevere pain are also narcotic agents and their use entails the risk ofproducing physical dependence. There are as yet no agents effectiveagainst severe pain that are entirely free of this risk.

Thus, there is an urgent need for improved analgesic agents for treatingmild as well as severe pain. The present invention provides such agents.

In addition to the need for improved analgesic agents, there is also aneed for improved psychotropic agents to supplement or to provide analternative to current therapy. The compounds of this invention, inaddition to their analgesic activity, also exhibit anti-depressant,tranquilizing, sedative and hypnotic activity. Thus, their usefulness asanalgesic agents is enhanced, since many patients suffering from painalso exhibit varying states of anxiety and depression.

A recently identified pentapeptide, methionine enkephalin, has thefollowing structure H-Tyr-Gly-Gly-Phe-Met-OH [see Hughes et al., Nature,258, 577 (1975)]. This peptide is found in many areas of the brain whereit appears to act as a neurotransmitter or neuromodulator in a centralpain suppressive system. The natural peptide binds stereo-specificallyto partially purified brain opiate receptor sites [for instance, seeBradbury et al., Nature, 260, 793 (1976)], is very active in bioassaysfor opiate activity, but exhibits only weak analgesic agent of shortduration when injected directly into the brain of the rat, [forinstance, see Belluzzi et al., Nature, 260, 625 (1976)].

In addition, several C-terminal fragments of a 91 chain length peptide,human camel and porcine β-lipotropin having the pentapeptide sequence ofmethionine enkephalin at their N-terminus have been isolated from thepituitory and found to exhibit potent opioid activity in binding topartially purified brain opiate receptor sites. See Ling and Guillemin,Proc. Natl. Acad. Sci USA 73, 3308 (1976) and Proc. Nat. Acad. Sci. USA73, 1821 (1976). The reported fragments have been characterized asα-endorphin (human β-lipotropin fragment 61-76), β-endorphin (humanβ-lipotropin fragment 61-91), γ-endorphin (human β-lipotropin fragment61-77) and δ-endorphin (human β-lipotropin fragment 61-87). Unlike theenkephalins, the endorphins possess some pharmacological activity whenadministered either intra-cerebrally or parenterally. The presentinvention is directed to novel derivatives of human β-endorphin whichare highly active by a variety of routes of administration and areuseful as pharmacological agents. In addition, the compounds haveveterinary utility because of their potent prolactin releasing andgrowth harmone releasing activities.

We have unexpectedly found that the incorporation of a D-amino acid,such as D-alanine for the penultimate glycine residue at the N-terminusof α-endorphin greatly enhances the analgesic activity of thesecompounds. In addition to their analgesic activity, the compounds arealso useful as anti-depressant, sedative-hypnotic, and tranquilizingagents.

SUMMARY OF THE INVENTION

This invention relates to novel peptides, and more specifically relatesto novel β-endorphin derivatives of human β-lipotropin fragment 61-91which are useful as analgesic, tranquilizer, sedative, hypnotic andanti-depressant agents, as well as prolactin releasing and growthhormone releasing agents, to intermediates useful in the preparation ofthe novel pentapeptides, and to pharmaceutical compositions and methodsemploying such novel pentapeptides.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The compounds of this invention are hentriacontapeptides having thefollowing amino acid sequence represented by formula I:

    H-Tyr-X-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Gln-Y

wherein X is a chiral residue of a D-amino acid selected from the groupconsisting of D-alanine, D-leucine, D-isoleucine, D-valine,D-phenylalanine, D-tyrosine, D-tryptophan, D-serine, D-threonine,D-methionine, D-glutamic acid, D-glutamine, D-aspartic acid,D-asparagine, D-lysine, D-proline, D-histidine and D-arginine; Y isselected from the group consisting of hydroxy, amino, loweralkylamino,lowerdialkylamino, and lower alkoxy; and the pharmaceutically acceptablesalts thereof.

All chiral amino acid residues identified herein are in the natural orL-configuration unless otherwise specified.

In keeping with standard peptide nomenclature, abbreviations for chiralamino acid residues have been used herein as follows:

    ______________________________________                                        His - L-histidine  D-His - D-histidine                                        Tyr - L-tyrosine   Ile - L-isoleucine                                         D-Tyr - D-tyrosine D-Ile - D-isoleucine                                       Gly - glycine      Leu - L-leucine                                            Phe - L-phenylalanine                                                                            D-Leu - D-leucine                                          D-Phe - D-phenylalanine                                                                          Thr - L-threonine                                          Met - L-methionine D-Thr - D-threonine                                        D-Met - D-methionine                                                                             Val - L-valine                                             Ala - L-alanine    D-Val - D-valine                                           D-Ala - D-alanine  Pro - L-proline                                            Ser - L-serine     D-Pro - D-proline                                          D-Ser - D-serine   Gln - L-glutamine                                          Lys - L-lysine     D-Gln - D-glutamine                                        D-Lys - D-lysine   Glu - L-glutamic acid                                      Asn - L-asparagine D-Glu - D-glutamic acid                                    D-Asn - D-asparagine                                                                             Trp - L-tryptophan                                         D-Asp - D-aspartic acid                                                                          D-Trp - D-tryptophan                                       Asp - aspartic acid                                                           ______________________________________                                    

The term "pharmaceutically acceptable salts," as used herein, refers tothe non-toxic alkali metal, alkaline earth metal and ammonium saltscommonly used in the pharmaceutical industry including the sodium,potassium, lithium, calcium, magnesium, barium and ammonium salts whichare prepared by methods well known in the art. The term also includesnon-toxic acid addition salts which are generally prepared by reactingthe compounds of this invention with a suitable organic or inorganicacid. Representative salts include the hydrochloride, hydrobromide,sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, napsylate, and the like.

The term "lower alkyl" refers to straight and branched chain alkylgroups having from 1 to 6 carbon atoms such as methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl and thelike.

The term "lower alkoxy" refers to straight and branched chain alkoxygroups having from 1 to 6 carbon atoms such as methoxy, ethoxy, and thelike.

Also contemplated within the scope of the present invention are thefollowing intermediates which are useful in preparing thehentriacontapeptides of this invention and are represented by formulaII.

    R.sub.1 -Tyr(R.sub.2)-X-Gly-Phe-Met-Thr(R.sub.3)-Ser(R.sub.3)-Glu(R.sub.4)-Lys(R.sub.5)-Ser(R.sub.3)-Gln-Thr(R.sub.3)-Pro-Leu-Val-Thr(R.sub.3)-Leu-Phe-Lys(R.sub.5)-(II)

Asn-Ala-Ile-Ile-Lys(R₅)-Asn-Ala-Tyr(R₂)-Lys(R₅)-Lys(R₅)-Gly-Gln-Y

wherein X is as defined in Formula I or, when X is tyrosine, threonine,serine, glutamic acid or lysine, a protected chiral residue as definedbelow; R₁ is a protecting group which would be used by one skilled inthe art in solid phase peptide synthesis selected from the groupconsisting of acyl type protecting groups, aromatic urethan-typeprotecting groups, alkyl type protecting groups, trialkylsilane groups,or aliphatic urethan protecting groups;

R₂ is a protecting group for the phenolic hydroxyl group of tyrosineselected from the group consisting of tetrahydropyranyl tert-butyl,trityl, benzyl, 2,4-dichlorobenzyl, benzyloxycarbonyl or2-bromobenzyloxycarbonyl (2-Br-Z);

R₃ is a protecting group for the alcohol hydroxy functions of serine andthreonine and is selected from the group defined hereinbefore for R₂ ;

R₄ is protecting group for the gamma carboxyl group of glutamic acidselected from the group consisting of tert-butyl or 4-chlorobenzyl;

R₅ is a protecting group for the epsilon amino group of lysine selectedfrom the group consisting of trifluoroacetyl, benzyloxycarbonyl or,preferably, 2-chlorobenzyloxycarbonyl; and

Y is as defined in Formula I or a derivatized insoluble polystyreneresin support represented by Formulas III or IV: ##STR1##

The term "acyl type protecting groups" refers to groups illustrated bybut not restricted to formyl, trifluoroacetyl, tosyl, nitrosulfonyl, andthe like.

The term "aromatic urethan-type protecting groups" is represented bygroups such as benzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-biphenylisopropyloxycarbonyl,2,5-dimethyoxyphenylisopropyloxycarbonyl, and the like.

The term "cycloalkyl urethan protecting group", as used herein, refersto groups such as cyclopentyloxycarbonyl, adamantyloxycarbonyl,cyclohexyloxycarbonyl, isoboronyloxycarbonyl, etc.

"Alkyl type protecting groups" are those commonly used in the art suchas the trityl group.

"Trialkylsilane groups" include compounds such as trimethylsilane,triethylsilane, tributylsilane, and the like.

The preferred protecting groups, the "aliphatic urethan protectinggroups", include tert-butyloxycarbonyl, diisopropyloxycarbonyl,isopropyloxycarbonyl, allyloxycarbonyl, and the like.

The polystyrene resin support is preferably a copolymer of styrene with1-2 weight percent of divinyl benzene as a crosslinking agent whichcauses the polystyrene polymer to be completely insoluble in mostorganic solvents. In Formula IV, φ is phenyl.

In selecting a particular side-chain protecting group to be used in thesynthesis of the peptides several conditions must be met: (a) theprotecting group must be stable to the reagent and under reactionconditions selected for removing the α-amino protecting group at eachstep of the synthesis; (b) the protecting group must retain itsprotecting properties and not be chemically modified; and (c) theside-chain protecting group must be removable at the end of the solidphase synthesis under reaction conditions that will not alter thepeptide chain.

The peptides are prepared using standard solid-phase techniques. Thesynthesis is commenced from the C-terminal end of the peptide using aα-amino protected resin. A suitable starting material can be prepared,for instance, by attaching the required α-amino acid to achloromethylated resin, a hydroxymethyl resin, or a benzhydrylamineresin. One such chloromethylated resin is sold under the tradenameBio-beads SX-1 by Bio Rad Laboratories, Richmond, Calif., and thepreparation of the hydroxymethyl resin is described in Bodanszky et al.,Chem. Ind. (London) 38, 1597 (1966). The benzhydrylamine resin has beendescribed by Pietta and Marshall, Chem. Commun., 650 (1970) and iscommercially available from Beckman Instrument, Palo Alto, Calif.

In the preparation of the compounds of this invention, an α-aminoprotected amino acid is coupled to the chloromethylated resin with theaid of, for example, cesium bicarbonate catalyst, according to themethod described by Gisin, Helv. Chim. Acta, 56, 1476 (1973). After theinitial coupling, the α-amino protecting group is removed by a choice ofreagents including trifluoroacetic acid or hydrochloric acid solutionsin organic solvents at room temperature. After removal of the α-aminoprotecting group, the remaining protected amino acids are coupledstepwise in the desired order to obtain the compounds of Formula II.Each protected amino acid is generally reacted in a 3-fold excess usingan appropriate carboxyl group activator such as dicyclohexylcarbodiimidein solution in, for example, methylene chloride-dimethylformamidemixtures.

After the desired amino acid sequence has been completed, the desiredpeptide is removed from the resin support by treatment with a reagentsuch as hydrogen fluoride which not only cleaves the peptide from theresin, but also cleaves all remaining side-chain protecting groups. Whenthe chloromethylated resin is used, hydrogen fluoride treatment resultsin the formation of the free peptide acids of Formula I (Y=OH). When thebenzhydrylamine resin is used, hydrogen fluoride treatment resultsdirectly in the free peptide amides of Formula I (Y--NH₂).Alternatively, when the chloromethylated resin is employed, theside-chain protected peptide can be cleaved by treatment of thepeptide-resin with ammonia to give the desired side-chain protectedamide or with an alkylamine or dialkylamine to give a side-chainprotected alkylamide or dialkylamide. Side-chain protection is thenremoved in the usual fashion by treatment with hydrogen fluoride to givethe free amides, alkylamides or dialkylamides.

In preparing the esters of this invention (Y=lower alkoxy), the resinused to prepare the acids of Formula I (Y=OH) is employed and thepeptide is removed from the resin by treatment with base and theappropriate alcohol, i.e., methanol. Sidechain protection is thenremoved in the usual fashion by treatment with hydrogen fluoride toobtain the desired ester.

The solid-phase procedure discussed above is well known in the art andhas been essentially described by J. M. Stewart, Solid Phase PeptideSynthesis (Freeman and Co., San Francisco, 1969).

The compounds of Formula I are useful as analgesics, anti-depressants,tranquilizers, sedatives or hypnotics when administered to mammalianhosts at dosages of from 0.001 to 100 mg/kg of body weight daily,preferably in divided dosages. The compounds are preferably administeredby parenteral routes, i.e., the intravenous, intraperitoneal,intramuscular or subcutaneous routes of administration, but may also beadministered by a variety of other routes including the oral orsublingual, or by vaginal, rectal or nasal routes of administration.Accordingly, one aspect of the present invention includes pharmaceuticalcompositions suitable for such routes of administration.

The analgesic activity of the compounds of Formula I was established inthe rat tail flick test as described by D'Amour and Smith, J. Pharmac.Exp. Ther., 72, 74 (1941). The presently preferred analgesic agent ofthis invention is D-Ala² -human β-lipotropin fragment (61-91) (alsoknown as D-Ala² -human β-endorphin).

The anti-depressant (stimulant) activity and the tranquilizing andsedative-hypnotic activity of the compounds of Formula I were firstestablished in the open field test described by Kulkarni et al.Pharmakopsychiatrie Neuro-Psychopharamakologie 8(1): pp. 45-50 (1975)and the self stimulation test described by Bailey et al., ResearchCommunications in Chemical Pathology and Pharmacology 11(4): pp. 543-552(1975).

The dosage administered depends upon the desired effect. For example,the degree of analgesia produced by the compounds of this invention maybe varied by varying the dosage. Anti-depressant activity is observed atthe lower dosages, i.e., 0.001 to 5 mg/kg and sedation ortranquilization is produced by dosages of greater than 5 mg./kg. of bodyweight.

The following compounds are illustrative of the hexadecapeptides ofFormula I. Compounds of Formula I will hereinafter be designated asderivatives of β-endorphin. The D-amino acid is designated as being inthe 2-position. Compounds wherein Y- is OH are named as substitutedβ-endorphins and are illustrated by the following:

D-Ala² -β-endorphin;

D-Leu² -β-endorphin;

D-Ile² -β-endorphin;

D-Val² -β-endorphin;

D-Phe² -β-endorphin;

D-Tyr² -β-endorphin;

D-Trp² -β-endorphin;

D-Ser² -β-endorphin, sodium salt;

D-Thr² -β-endorphin;

D-Met² -β-endorphin; calcium salt;

D-Glu² -β-endorphin;

D-His² -β-endorphin;

D-Pro² -β-endorphin

D-Gln² -β-endorphin; ammonium salt;

D-Asp² -β-endorphin;

D-Asn² -β-endorphin;

D-Lys² -β-endorphin;

D-Arg² -β-endorphin;

The endorphin amides of this invention include but are not limited tothe following:

D-Ala² -β-endorphin amide;

D-Leu² -β-endorphin amide;

D-Ile² -β-endorphin amide;

D-Val² -β-endorphin amide;

D-Phe² -β-endorphin amide;

D-Tyr² -β-endorphin amide;

D-Trp² -β-endorphin amide;

D-Ser² -β-endorphin amide;

D-Thr² -β-endorphin amide;

D-Met² -β-endorphin amide;

D-Glu² -β-endorphin amide;

D-Gln² -β-endorphin amide;

D-Asp² -β-endorphin amide;

D-Asn² -β-endorphin amide;

D-Lys² -β-endorphin amide;

D-Arg² -β-endorphin amide;

D-Ala² -β-endorphin amide hydrochloride;

D-Leu² -β-endorphin amide citrate;

D-Ile² -β-endorphin amide hydrobromide;

D-Val² -β-endorphin amide hydroiodide;

D-Phe² -β-endorphin amide hydrochloride;

D-Tyr² -β-endorphin amide sulfate;

D-Trp² -β-endorphin amide lactate;

D-Ser² -β-endorphin amide napsylate;

D-Pro² -β-endorphin amide;

D-His² -β-endorphin amide;

D-Thr² -β-endorphin amide oleate;

D-Met² -β-endorphin amide valerate;

D-Glu² -β-endorphin amide tosylate;

D-Gln² -β-endorphin amide disulfate;

D-Asp² -β-endorphin amide benzoate;

D-Asn² -β-endorphin amide acetate;

D-Lys² -β-endorphin amide laurate;

D-Arg² -β-endorphin amide phosphate;

D-Ala² -β-endorphin methyl amide;

D-Leu² -β-endorphin ethyl amide;

D-Ile² -β-endorphin n-propyl amide;

D-Val² -β-endorphin n-butyl amide;

D-Phe² -β-endorphin tert-butyl amide;

D-Tyr² -β-endorphin sec-butyl amide;

D-Trp² -β-endorphin n-pentyl amide;

D-Ser² -β-endorphin ethyl amide;

D-Thr² -β-endorphin dimethyl amide;

D-Met² -β-endorphin diethyl amide;

D-Glu² -β-endorphin n-propyl amide;

D-Gln² -β-endorphin-iso-propyl amide; and the like.

Esters of this invention include but are not limited to the following:

D-Ala² -β-endorphin, methyl ester;

D-Val² -β-endorphin, ethyl ester;

D-Met² -β-endorphin, n-propyl ester;

D-Phe² -β-endorphin, iso-propyl ester;

D-Glu² -β-endorphin, n-butyl ester;

D-Gln² -β-endorphin, n-pentyl ester;

D-Ser² -β-endorphin, n-hexyl ester; and the like.

The following examples further illustrate the present invention:

EXAMPLE 1

Preparation ofO-2-bromobenzyloxycarbonyl-L-tyrosyl-D-alanyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-lucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylallanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzylloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin

Tert-butyloxycarboxyl-gamma-benzyl-glutamic acid-O-CH₂ -resin (2.16 g,1.0 mmole), prepared by the method of Gisin, Helv. Chim. Acta, 56, 1476(1973), is placed in the reaction vessel of a Beckman Model 990automatic peptide synthesizer programmed to carry out the followingcycle of washes and reactions: (a) methylene chloride; (b) 25%trifluoroacetic acid in methylene chloride (2 times for 1.5 and 15minutes each); (c) 50% trifluoroacetic acid in methylene chloride (15minutes); (d) methylene chloride; (e) ethanol; (f) methylene chloride;(g) 10% triethylamine in methylene chloride (2 times for 5 minuteseach); and (h) methylene chloride.

The deprotected resin is then stirred with tert-butyloxycarbonyl (t-Boc)glycine (3.0 mmoles) in methylene chloride and dicyclohexylcarbodiimide(3.0 mmoles) is added thereto. The mixture is stirred at roomtemperature for 2 hours and the peptide resin is then washedsuccessively with methylene chloride, ethanol and methylene chloride.Two percent N-acetylimidazole in metylene chloride is then added and themixture stirred for 15 minutes in order to irreversibly acetylateunreacted free amino groups. The resin is then washed with methylenechloride followed by ethanol and then steps (a) through (h) are repeatedas described above.

The remaining 29 Boc-amino acids are then coupled successively by thesame cycle of events and the completed peptide-resin is washed withmethanol (3 times) and dried under reduced pressure whereupon 5.64 g. ofmaterial is obtained.

EXAMPLE 2

Preparation ofL-tyrosyl-D-alanyl-glycyl-L-phenylalanyl-L-methionyl-L-threonyl-L-seryl-L-glutamyl-L-lysyl-L-seryl-L-glutamyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-L-threonyl-L-leucyl-L-phenylalanyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-L-lysyl-L-asparagiyl-L-alanyl-L-tyrosyl-L-lysyl-L-lysyl-glycyl-L-glutamic acid (D-Ala²-β-endorphin)

Removal of the protecting groups and cleavage of the peptide from theresin obtained according to the method of Example 1 is carried out bytreating 1.45 g. of the peptide-resin with hydrogen fluoride (40 ml) andanisole (4 ml) at 0° for 45 minutes. The hydrogen fluoride is removedunder reduced pressure and the anisole removed by washing with ethylacetate.

The crude peptide is purified by gel filtration on a column (2.5×95 cm)of Sephadex G50 by elution with 2 molar acetic acid and fractions shownto contain a major peak by uv absorption at 280 nm are pooled andevaporated to dryness. The residual oil is applied to a column (2.5×95cm) of Sephadex G50 gel previously equilibrated with the lower phasefollowed by the upper phase of a 0.1% acetic acid: n-butanol:pyridine(11:5:3) solvent system. Elution with the upper phase yields a majorsymmetrical peak, determined by Folin-Lowoy measurements, and materialfrom this area is evaporated by dryness and lyophilized from water toyield 85 mg of white powder. This is eluted on a column (1.5×45 cm) ofcarboxymethyl-cellulose with a linear gradient of ammonium acetatebuffer (0.1 M, pH 4.6 to 0.4 M, pH 7.0) and the major symmetrical peak,as determined by uv absorption, yields 55 mg of white powder afterrepeated lyophilization.

The product is homogeneous by thin layer chromatography in 5 solventsystems on silica gel plates when loads of 30 ug are applied andvisualized by exposure to ninhydrin reagent followed bychlorine/starch-potassium iodide reagents. The following Rf values wereobtained:

(A) n-Butanol:acetic acid:water (4:1:5 upper phase), 0.01;

(B) ethyl acetate:pyridine:acetic acid:water (5:5:1:3), 0.36;

(C) i-proporol:1 Moretic acid (2:1), 0.31;

(D) n-butanol:acetic acid:water:ethyl acetate (1:1:1:1), 0.29;

(E) n-butanol: pyridine:acetic acid:water (15:10:3:12), 0.32.

EXAMPLE 3

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-leucyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-leucine derivative instead of the t-Boc-D-alanine derivative.

EXAMPLE 4

D-Leu² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 3.

EXAMPLE 5

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-isoleucyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-N-epsilon-2-chlorobenxyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-isoleucine, instead of t-Boc-D-alanine.

EXAMPLE 6

D-Ile² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 5.

EXAMPLE 7

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-valyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-valine, instead of t-Boc-D-alanine.

EXAMPLE 8

D-Val² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 7.

EXAMPLE 9

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-phenylalanylglycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-phenylalanine derivative instead of the t-Boc-D-alaninederivative.

EXAMPLE 10

D-Phe² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 9.

EXAMPLE 11

0-2-Bromobenzyloxycarbonyl-L-tyrosyl-2-bromobenzyloxycarbonyl-D-tyrosyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-tyrosine derivative instead of t-Boc-D-alanine.

EXAMPLE 12

D-Tyr² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 11.

EXAMPLE 13

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-tryptophanylglycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-tryptophan, instead of t-Boc-D-alanine.

EXAMPLE 14

D-Trp² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 13.

EXAMPLE 15

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-O-benzyl-D-serylglycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-serine derivative instead of t-Boc-D-alanine.

EXAMPLE 16

D-Ser² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 15.

EXAMPLE 17

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-O-benzyl-D-threonylglycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-threonine derivative instead of t-Boc-D-alanine.

EXAMPLE 18

D-Thr² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 17.

EXAMPLE 19

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-methionyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-propyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-methionine derivative instead of t-Boc-D-alanine.

EXAMPLE 20

D-Met² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 19.

EXAMPLE 21

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-gamma-benzyl-D-glutamyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-glutamic acid derivative instead of t-Boc-D-alanine.

EXAMPLE 22

D-Glu² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 21.

EXAMPLE 23

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-glutaminylglycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonylL-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH.sub.2-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-glutamine derivative instead of t-Boc-D alanine.

EXAMPLE 24

D-Gln² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 23.

EXAMPLE 25

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-alpha-benzyl-D-aspartyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-aspartic acid derivative instead of t-Boc-D-alanine.

EXAMPLE 26

D-Asp² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 25.

EXAMPLE 27

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-asparaginylglycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-asparagine derivative instead of t-Boc-D-alanine.

EXAMPLE 28

D-Asn² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 27.

EXAMPLE 29

O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-D-lysyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-lysine derivative instead of t-Boc-D-alanine.

EXAMPLE 30

D-Lys² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 29.

EXAMPLE 31

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-N-guanidino-tosyl-D-arginyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-O-CH₂-resin is prepared by the method of Example 1, using the correspondingt-Boc-D-arginine derivative instead of t-Boc-D-alanine.

EXAMPLE 32

D-Arg² -β-endorphin is prepared by the method of Example 2, from thepeptide resin of Example 31.

EXAMPLE 33

Preparation ofO-2-bromobenzyloxycarbonyl-L-tyrosyl-D-alanyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin

Using the conditions described in Example 1, the t-Boc derivatives ofeach amino acid are successively coupled to a benzhydrylamine resin(0.63 g, 0.5 mmole), purchased from Beckman Instruments, Palo Alto,Calif.

EXAMPLE 34

D-Ala² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 33.

EXAMPLE 35

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-leucyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-leucine, instead of t-Boc-D-alanine.

EXAMPLE 36

D-Leu² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 35.

EXAMPLE 37

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-isoleucyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-isoleucine, instead of t-Boc-alanine.

EXAMPLE 38

D-Ile² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 37.

EXAMPLE 39

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-valyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-valine, instead of t-Boc-D-alanine.

EXAMPLE 40

D-Val² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 39.

EXAMPLE 41

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-phenylalanylglycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyk-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-bezhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-phenylalanine, instead of t-Boc-D-alanine.

EXAMPLE 42

D-Phe² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 41.

EXAMPLE 43

O-2-Bromobenzykoxycarbonyl-L-tyrosyl-2-bromobenzyloxycarbonyl-D-tyrosyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxyca-bonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-tyrosine derivative instead of t-Boc-D-alanine.

EXAMPLE 44

D-Tyr² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 43.

EXAMPLE 45

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-tryptophanylglycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamylbenzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-tryptophan, instead of t-Boc-D-alanine.

EXAMPLE 46

D-Trp² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 45.

EXAMPLE 47

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-O-benzyl-D-seryl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-serine derivative instead of t-Boc-D-alanine.

EXAMPLE 48

D-Ser² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 47.

EXAMPLE 49

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-O-benzyl-L-threonyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenxyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-threonine derivative instead of t-Boc-D-alanine.

EXAMPLE 50

D-Thr² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 49.

EXAMPLE 51

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-methionyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilin-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-N-epsilon-2-chlorobensyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamylbenzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-methionine derivative, instead of t-Boc-D-alanine.

EXAMPLE 52

D-Met² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 51.

EXAMPLE 53

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-gamma-benzyl-D-glutamyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-glutamic acid derivative, instead of t-Boc-D-alanine.

EXAMPLE 54

D-Glu² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 53.

EXAMPLE 55

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-glutaminyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valvl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-glutamine, instead of t-Boc-D-alanine.

EXAMPLE 56

D-Gln² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 55.

EXAMPLE 57

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-alpha-benzyl-D-aspartyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alynl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorocarbobenzyloxy-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-aspartic acid derivative, instead of t-Boc-D-alanine.

EXAMPLE 58

D-Asp² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 57.

EXAMPLE 59

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-D-asparaginyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonal-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-asparagine, instead of t-Boc-D-alanine.

EXAMPLE 60

D-Asn² -β-endorphin is prepared by the method of Example 2 from thepeptide resin of Example 59.

EXAMPLE 61

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-D-lysyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl-L-prolyl-L-leucyl-L-valyl-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-lysine, instead of t-Boc-D-alanine.

EXAMPLE 62

D-Lys² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 61.

EXAMPLE 63

O-2-Bromobenzyloxycarbonyl-L-tyrosyl-N-guanidino-tosyl-D-arginyl-glycyl-L-phenylalanyl-L-methionyl-O-benzyl-L-threonyl-O-benzyl-L-seryl-gamma-benzyl-L-glutamyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-O-benzyl-L-seryl-L-glutaminyl-O-benzyl-L-threonyl1-prolyl-L-leucyl-L-valylo-O-benzyl-L-threonyl-L-leucyl-L-phenylalanyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-L-isoleucyl-L-isoleucyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-L-asparaginyl-L-alanyl-O-2-bromobenzyloxycarbonyl-L-tyrosyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-N-epsilon-2-chlorobenzyloxycarbonyl-L-lysyl-glycyl-gamma-benzyl-L-glutamyl-benzhydrylamineresin is prepared by the method of Example 1, using the correspondingt-Boc-D-arginine derivative, instead of t-Boc-D-alanine.

EXAMPLE 64

D-Arg² -β-endorphin amide is prepared by the method of Example 2 fromthe peptide resin of Example 63.

Examples 65-73 illustrate the preparation of the esters of thisinvention. It will be readily apparent to one skilled in the art thatesters of a D-amino acid² substituted endorphin are prepared by reactingthe peptide resins of Examples 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,23, 25, 27, 29, 31, 33 and 35 with the appropriate alcohol in thepresence of base and then proceeding as in Example 2 with hydrogenfluoride treatment and subsequent purification of the desired material.

EXAMPLE 65

The methyl ester of D-Ala² -β-endorphin is prepared from the resin ofExample 1 by cleaving the peptide from the resin with ethanol (50 ml) inthe presence of triethylamine (40 mmoles). Removal of the protectinggroups is carried out according to the method of Example 2 by treatingthe peptide with hydrogen fluoride.

EXAMPLE 66

The ethyl ester of D-Leu² -β-endorphin is prepared by the method ofExample 65 from the resin of Example 3, using ethanol in place ofmethanol and conducting the reaction at elevated temperatures.

EXAMPLE 67

The n-propyl ester of D-Ile² -β-endorphin is prepared by the method ofExample 66 from the resin of Example 5, using n-propanol instead ofethanol.

EXAMPLE 68

The isopropyl ester of D-Val² -β-endorphin is prepared from the resin ofExample 7, by the method of Example 66, using isopropanol in place ofethanol.

EXAMPLE 69

The n-butyl ester of D-Phe² -β-endorphin is prepared from the resin ofExample 9, by the method of Example 66, using the n-butanol instead ofethanol.

EXAMPLE 70

The tert-butyl ester of D-Tyr² -β-endorpin is prepared from the resin ofExample 11 by the method of Example 66, using tert-butanol instead ofethanol.

EXAMPLE 71

The sec-butyl ester of D-Trp² -β-endorphin is prepared from the resin ofExample 13 by the method of Example 66, using the sec-butanol instead ofethanol.

EXAMPLE 72

The n-pentyl ester of D-Ser² -β-endorphin is prepared from the resin ofExample 15 by the method of Example 66, using the n-pentanol instead ofethanol.

EXAMPLE 73

The n-hexyl ester of D-Thr² -β-endorphin is prepared from the resin ofExample 17 by the method of Example 66, using n-hexanol instead ofethanol.

The following examples are illustrative of the preparation of thealkylamides and dialkyamides of this invention.

EXAMPLE 74

D-Ala² -β-endorphin methyl amide is prepared by reacting the peptideresin of Example 1 with a large excess of methylamine indimethylformamide followed by removel of the side-crain protectinggroves of the cleaved peptide methylamide by treatment with hydrogenfluoride in the presence of anisole under the standard conditions.

EXAMPLE 75

D-Leu² -β-endorphin ethyl amide is prepared from the peptide-resin ofExample 3 by the method of Example 74, using ethylamine in place ofmethylamine.

EXAMPLE 76

D-Ile² -β-endorphin, n-propyl amide is prepared from the peptide-resinof Example 5 by the method of Example 74, using n-propylamine in placeof methylamine.

The present invention includes within its scope pharmaceuticalcompositions comprising, as an active ingredient, at least one of thecompounds of formula I in association with a pharmaceutical carrier ordiluent. The compounds of this invention can be administered by oral,parenteral (intramuscular, intravenous or subcutaneous injection),nasal, vaginal, rectal or sublingual routes of administration and can beformulated in dosage forms appropriate for each route of administration.

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, the activecompound is admixed with at least one inert diluent such as sucrose,lactose, or starch. Such dosage forms can also comprise, as is normalpractice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water.Besides such inert diluents, compositions can also include adjuvants,such as wetting agents, emulsifying and suspending agents, and sweeting,flavoring, and perfuming agents.

Preparations according to this invention for parenteral administrationinclude sterile aqueous or non-aqueous solutions, suspensions, oremulsions. Examples of non-aqueous solvents or vehicles are propyleneglycol, polyethylene glycol, vegetable oils, such as olive oil, andinjectable organic esters such as ethyl oleate. Such dosage forms mayalso contain adjuvants such as preserving, wetting, emulsifying, anddispersing agents. They may be sterilized by, for example, filtrationthrough a bacteria-retaining filter, by incorporating sterilizing agentsinto the compositions, by irradiating the compositions, or by heatingthe compositions. They can also be manufactured in the form of sterilesolid compositions which can be dissolved in sterile water, or someother sterile injectable medium immediately before use.

Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as cocoa butter or a suppository wax.

Compositions for nasal or sublingual administration are also preparedwith standard excipients well known in the art.

The dosage of active ingredient in the compositions of this inventionmay be varied; however, it is necessary that the amount of the activeingredient shall be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment.Generally, dosage levels of between 0.001 to 100 mg/kg. of body weightof daily are administered to mammals to obtain effective relief frompain or to relieve depression.

The following examples further illustrate the pharmaceuticalcompositions which are a feature of this invention.

EXAMPLE 77

Tablets weighing 200 mg. and having the following compositions areformulated:

    ______________________________________                                        Ingredient        Mg                                                          ______________________________________                                        D-Ala.sup.2 -β-endorphin                                                                   50                                                          Starch            120                                                         Colloidal silica  27                                                          Magnesium stearate                                                                               3                                                          ______________________________________                                    

EXAMPLE 78

Sterile 10 ml. ampules can be prepared containing 10 mg per ml of D-Ala²-β-endorphin, ethyl ester 0.1 percent sodium bisulfate, 0.7 percentsodium chloride, and 0.5 percent chlorobutanol as a preservative.

EXAMPLE 79

Topical aqueous formulations for administration by nose drops or nasalspray are formulated containing 1 mg. of D-Ile² -β-endorphin amide, 3.8mgm glycerine, 40 mg. sorbital, 0.02 mg. benzalkonium chloride andpurified water g.s. 1 m.

We claim:
 1. Hentriacospeptides represented by the formula

    H-Tyr-X-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn-Ala-Ille-Ille-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu-Y

wherein X is a chiral residue of a D-amino acid selected from the groupconsisting of D-leucine and D-lysine; Y is selected from the groupconsisting of hydroxy, amino, loweralkylamino, lower dialkylamino andlower alkoxy; and the pharmaceutically acceptable salts thereof.
 2. Thehentriacosapeptides of claim 1 wherein Y is amino.
 3. Thehentriacosapeptides of claim 1 wherein Y is loweralkylamino.
 4. Thehentriacosapeptides of claim 1 wherein Y is lower dialkylamino.
 5. Thehentriacosapeptides of claim 1 wherein Y is lower alkoxy.
 6. Apharmaceutical composition suitable for oral, parenteral, nasal, rectalor vaginal or sublingual administration comprising a therapeuticallyeffective amount of a compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 7. A pharmaceutical composition inaccordance with claim 6 adapted for oral administration.
 8. Apharmaceutical composition in accordance with claim 6 adapted forparenteral administration.
 9. A pharmaceutical composition in accordancewith claim 6 adapted for nasal administration.
 10. A parmaceuticalcomposition in accordance with claim 6 adapted for rectaladministration.
 11. A pharmaceutical composition in accordance withclaim 6 adapted for vaginal administration.
 12. A pharmaceuticalcomposition in accordance with claim 6 adapted for sublingualadministration.
 13. A method of relieving pain in a mammalian patientsuffering therefrom comprising administering a therapeutically effectiveamount of a compound of claim 1 to said patient.
 14. A method oftreating a mammalian patient exhibiting the signs of depressioncomprising, administering a therapeutically effective amount of acompound of claim 1 to said patient.
 15. A method of sedating amammalian patient in need of such treatment comprising, administering atherapeutically effective amount of a compound of claim 1 to saidpatient.
 16. A method of tranquilizing a mammalian patient in need ofsuch treatment comprising, administering a therapeutically effectiveamount of a compound of claim 1 to said patient.
 17. Ahentriacosapeptide of the formula

    R.sub.1 -Tyr(R.sub.2)-X(Z)--Gly-Phe-Met-Thr(R.sub.3)-Ser(R.sub.3)-Glu(R.sub.4)-Lys(R.sub.5)-Ser(R.sub.3)-Gln-Thr(R.sub.3)-Pro-Leu-Val-Thr(R.sub.3)-Leu-Phe-Lys(R.sub.5)-Asn-Ala-Ile-Ile-Lys(R.sub.5)-Asn-Ala-Tyr(R.sub.2)-Lys(R.sub.5)-Lys(R.sub.5)-Gly-Gln(R.sub.4)-Y

wherein X is a chiral residue of a D-amino acid selected from the groupconsisting of D-leucine and D-lysine; Z is a protecting group for thechiral residue, M is an integer from zero to one, being one when X isD-lysine and being zero for any other residue; R₁ is a N-terminus solidphase peptide synthesis protecting group selected from the groupconsisting of acyl type protecting groups, aromatic urethan-typeprotecting groups, alkyl type protecting groups, trialkylsilane groups,or aliphatic urethan protecting groups; R₂ is a protecting group for thephenolic hydroxyl group of tyrosine selected from the group consistingof tetrahydropyranyl, tert-butyl, trityl, benzyl, 2,4-dichlorobenzyl,benzyloxycarbonyl and 2-bromobenzyloxycarbonyl; R₃ is a protecting groupfor the alcohol hydroxy functions of serine and threonine; R₄ is aprotecting group for the gamma carboxyl group of glutamic acid; R₅ is aprotecting group for the epsilon amino group of lysine selected from thegroup consisting of trifluoraacetyl, benzyloxycarbonyl, and2-chlorobenzyloxycarbonyl; and Y is selected from the group consistingof hydroxy, amino, loweralkylamino, diloweralkylamino, and lower alkoxy;or a derivatized insoluble polystyrene resin support represented by theformulas ##STR2##
 18. A β-endorphin analog selected from the groupconsisting of [D-Leu² ] and [D-Lys² ]-β-endorphin.
 19. Thehentriacosapeptides of claim 1, wherein X is a chiral residue ofD-leucine.
 20. The hentriacosapeptides according to claim 1 wherein X isa chiral residue of D-lysine.